This invention relates to fluorescence microscopy in which magnified images of tissues, cells or other components of a sample are obtained from light emission from a plurality of fluorescent dyes which can be attached to specific components or features of the sample.
High resolution imaging has and continues to play a prominent role in biology. Microscopic techniques have improved steadily in both the cellular and sub-cellular realms. Fluorescence microscopy is presently preferred because of its superior image contrast over that of conventional light scattering microscopy and because of the now well established procedure of attaching a wide variety of dyes to target related components which allows for high resolution visualization of structure-specific bindings resulting in rapid growth of an important sub-field of immunocytology.
Practical fluorescence microscopy is about fifty years old. In 1935, Max Hartinger first substituted fluorescent dyes for the then conventional stains used in histological procedures. This was developed into a routine procedure for bacteriology laboratories in the 1940's with the use of Acridine Orange as a metachromatic fluorescent stain for nucleic acids and the labeling of various proteins including specific antibodies, with isocyanate and isothiocyanate derivatives of fluorescein. Subsequently, an extensive series of other fluorescent agents, some of which are brighter and have greater avidity for specific targets, have been developed for cytological work. Technical advances such as monochromatic and epifluorescent illumination systems have reduced problems with dye bleaching and in some cases eliminated the need for barrier filters to screen out activating light.
The attractive possibility of using more than two fluorescent stains simultaneously in the same preparation, even when employing high contrast imaging techniques, has heretofore been unattainable because the spectral emission of the available dyes is typically very broad and emission in the blue range is a natural characteristic of all cells which masks or blanks out blue emitting dyes. Accordingly, only dyes that emit in the red and yellow ranges can be effectively distinguished even when employing available filtering techniques.
Use of time resolved fluorescence is an effective technique for interpreting dynamical interactions with the environment of the dye site and for obtaining information from non-single exponential decay modes. However, time resolved fluorescence has not been suggested or used for noninterfering particular spatial site selection with the exception of long decay time dyes for generalized background differentiation.
Fluorescent dyes are not the only possible site specific labels that can be used in association with present day imaging techniques. Others include shape and opacity defined labels such as latex particles, collodial gold and thorium particles, india ink, magnetite, erythrocytes and bacteria. Some of these serve purposes beyond imaging such as magnetite tagging which lends itself to separation processes and the like. In addition, there exist substrate imaging techniques such as insoluble substrate deposits and site specific alkaline etching, chemiluminescent stains and radioisotope stains which, although they provide imaging, are again limited to at most two labels simultaneously even when used in a high resolution system.